Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale.

BACKGROUND: The parasite species Plasmodium ovalecurtisi (P. ovalecurtisi) and Plasmodium ovalewallikeri (P. ovalewallikeri), formerly known as Plasmodium ovale, are endemic across multiple African countries. These species are thought to differ in clinical symptomatology and latency, but only a small number of existing diagnostic assays can detect and distinguish them. In this study, we sought to develop new assays for the detection and differentiation of P. ovalecurtisi and P. ovalewallikeri by leveraging recently published whole-genome sequences for both species. METHODS: Repetitive sequence motifs were identified in available P. ovalecurtisi and P. ovalewallikeri genomes and used for assay development and validation. We evaluated the analytical sensitivity of the best-performing singleplex and duplex assays using synthetic plasmids. We then evaluated the specificity of the duplex assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), and validated its performance using 55 P. ovale samples and 40 non-ovale Plasmodium samples from the DRC. RESULTS: The best-performing P. ovalecurtisi and P. ovalewallikeri targets had 9 and 8 copies within the reference genomes, respectively. The P. ovalecurtisi assay had high sensitivity with a 95% confidence lower limit of detection (LOD) of 3.6 parasite genome equivalents/µl, while the P. ovalewallikeri assay had a 95% confidence LOD of 25.9 parasite genome equivalents/µl. A duplex assay targeting both species had 100% specificity and 95% confidence LOD of 4.2 and 41.2 parasite genome equivalents/µl for P. ovalecurtisi and P. ovalewallikeri, respectively. CONCLUSIONS: We identified promising multi-copy targets for molecular detection and differentiation of P. ovalecurtisi and P. ovalewallikeri and used them to develop real-time PCR assays. The best performing P. ovalecurtisi assay performed well in singleplex and duplex formats, while the P. ovalewallikeri assay did not reliably detect low-density infections in either format. These assays have potential use for high-throughput identification of P. ovalecurtisi, or for identification of higher density P. ovalecurtisi or P. ovalewallikeri infections that are amenable to downstream next-generation sequencing.

Population Genomics of Plasmodium malariae from Four African Countries.

Plasmodium malariae is geographically widespread but neglected and may become more prevalent as P. falciparum declines. We completed the largest genomic study of African P. malariae to-date by performing hybrid capture and sequencing of 77 isolates from Cameroon (n=7), the Democratic Republic of the Congo (n=16), Nigeria (n=4), and Tanzania (n=50) collected between 2015 and 2021. There is no evidence of geographic population structure. Nucleotide diversity was significantly lower than in co-localized P. falciparum isolates, while linkage disequilibrium was significantly higher. Genome-wide selection scans identified no erythrocyte invasion ligands or antimalarial resistance orthologs as top hits; however, targeted analyses of these loci revealed evidence of selective sweeps around four erythrocyte invasion ligands and six antimalarial resistance orthologs. Demographic inference modeling suggests that African P. malariae is recovering from a bottleneck. Altogether, these results suggest that P. malariae is genomically atypical among human Plasmodium spp. and panmictic in Africa.

Population genomics of Plasmodium ovale species in sub-Saharan Africa.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) are relapsing malaria parasites endemic to Africa and Asia that were previously thought to represent a single species. Amid increasing detection of ovale malaria in sub-Saharan Africa, we performed a population genomic study of both species across the continent. We conducted whole-genome sequencing of 25 isolates from Central and East Africa and analyzed them alongside 20 previously published African genomes. Isolates were predominantly monoclonal (43/45), with their genetic similarity aligning with geography. Pow showed lower average nucleotide diversity (1.8×10-4) across the genome compared to Poc (3.0×10-4) (p < 0.0001). Signatures of selective sweeps involving the dihydrofolate reductase gene were found in both species, as were signs of balancing selection at the merozoite surface protein 1 gene. Differences in the nucleotide diversity of Poc and Pow may reflect unique demographic history, even as similar selective forces facilitate their resilience to malaria control interventions.

Longevity of Schistosoma mansoni circulating cathodic antigens in filter paper dried urine spots.

Objectives: This study aims to determine the temporal stability of Schistosoma mansoni circulating cathodic antigens (CCA) in filter paper-based dried urine spot (FP-DUS) samples under varying temperatures condition. Methods: Urine from 20 children confirmed to have S. mansoni infection using Kato-Katz (at least 1 egg per gram of stool) and Schisto POC-CCA (2+ and 3+) methods were stored in form of FP-DUS and urine at room temperature (RT), 4 °C and -20 °C. Standard urine and FP-DUS Schisto POC-CCA methods were employed to detect CCA in urine and FP-DUS samples respectively, at weeks 4, 8 and 12. The results were reported as negative or positive (trace, 1+. 2+, and 3+). Results: In FP-DUS samples, POC-CCA scores initially increase after 4-8 weeks, but then showed a decrease in intensity while still remaining positive, independent of temperature condition. From week 4 to week 12, at least 80 % of urine samples had POC-CCA score of 3+, independent of temperature condition. However, 2 urine samples at RT tested negative at weeks 8 and 12. Conclusions: Despite the decrease in the intensity of test line in many samples, S. mansoni CCA remains stable and detectable in urine samples stored in FP-DUS.

Efficacy and safety of artemether-lumefantrine against uncomplicated falciparum malaria infection in Tanzania, 2022, a single arm clinical trial.

BACKGROUND: Artemether-lumefantrine (AL) is the first line anti-malarial drug for the treatment of uncomplicated malaria in Tanzania. The World Health Organization (WHO) recommends regular efficacy monitoring of anti-malarial drugs to inform case management policy decisions. This study assessed the safety and efficacy of AL for treating uncomplicated P. falciparum malaria in Tanzania in 2022. METHODS: Children 6 months to 10 years with uncomplicated P. falciparum malaria were recruited from four sentinel sites and treated with the standard 6 dose, 3-day regimen for AL. Clinical and parasitological responses were monitored for 28 days using the WHO standard protocol. Genotyping based on msp1, msp2 and glurp was used to distinguish recrudescence from reinfection. SANGER sequencing was used to detect K13 mutations. RESULTS: 352 participants, 88 per site, were enrolled. Four withdrew and 55 experienced parasite recurrence. The PCR corrected Kaplan-Meier efficacies were, 89.9% in Pwani, 95.0% in Kigoma, 94.4% in Tanga, and 98.9% in Morogoro. No K13 mutations were found. CONCLUSIONS: Artemether-lumefantrine remains highly efficacious in three regions of Tanzania but the PCR-corrected efficacy in Pwani fell below the WHO-defined 90% threshold at which policy change is recommended. Implementing strategies to diversify ACTs to ensure effective case management in Tanzania is critical.

Urogenital schistosomiasis among adult male population in an endemic area of southern Tanzania: a descriptive cross-sectional study.

BACKGROUND: Urogenital schistosomiasis (UGS) caused by Schistosoma haematobium is endemic in Southern Tanzania. The disease has significant implications for both socioeconomic and public health. Because infections with S. haematobium usually peak in childhood, the majority of studies have concentrated on school-aged children leaving other groups such as males which might be continuous reservoir of infection transmission. However, despite its chronic consequences in the male population, the disease has received insufficient attention, especially in sub-Saharan Africa. This study was conducted to describe the previous and current schistosomiasis status among adult males living in high-endemic areas of southern Tanzania DESIGN, SETTING AND PARTICIPANTS: A descriptive cross-sectional study was employed to gather data on the prevalence of UGS among adult men residing at schistosomiasis endemic in the Mtama District Council. Quantitative methods of data collection which included questionnaire and laboratory procedures were used. RESULTS: Out of 245 participants, macrohaematuria and microhaematuria were found in 12 (4.9%, 95% CI 2.4% to 7.8%) and 66 (26.9%, 95% CI 21.6% to 32.7%) participants, respectively. S. haematobium ova were recovered from the urine samples of 54 (22.0%, 95% CI 16.7% to 27.3%) participants. The median intensity of infection was 20 eggs per 10 mL of urine ranging from 1 to 201 eggs per 10 mL of urine (IQR) 60.5). Out of 245 participants 33 (13.5% 95% CI 9.0% to 17.6%) had light intensity of infection and 21 (38.9%, 95% CI; 25.0% to 52.5%) had heavy intensity of infection. Overall, the prevalence of heavy intensity of infection was 8.6% (95% CI 4.9% to 12.6%). The prevalence and intensity of UGS varied significantly by age, marital status and village of residence. CONCLUSION: This study sheds light on the prevalence of UGS among adult males in endemic areas of southern Tanzania. The results highlight the urgent need for comprehensive intervention strategies to address the burden of the disease.

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

Background: The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania and continued local transmission. Methods: To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo district on the coastal mainland from 2016 to 2018. Results: Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to the rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low-level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Conclusions: Our data support importation as a main source of genetic diversity and contribution to the parasite population in Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive to malaria reemergence due to susceptible hosts and competent vectors. Funding: This research was funded by the National Institutes of Health, grants R01AI121558, R01AI137395, R01AI155730, F30AI143172, and K24AI134990. Funding was also contributed from the Swedish Research Council, Erling-Persson Family Foundation, and the Yang Fund. RV acknowledges funding from the MRC Centre for Global Infectious Disease Analysis (reference MR/R015600/1), jointly funded by the UK Medical Research Council (MRC) and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 program supported by the European Union. RV also acknowledges funding by Community Jameel.

Microheterogeneity of Transmission Shapes Submicroscopic Malaria Carriage in Coastal Tanzania.

BACKGROUND: Asymptomatic carriage of malaria parasites persists even as malaria transmission declines. Low-density infections are often submicroscopic, not detected with rapid diagnostic tests (RDTs) or microscopy but detectable by polymerase chain reaction (PCR). METHODS: To characterize submicroscopic Plasmodium falciparum carriage in an area of declining malaria transmission, asymptomatic persons >5 years of age in rural Bagamoyo District, Tanzania, were screened using RDT, microscopy, and PCR. We investigated the size of the submicroscopic reservoir of infection across villages, determined factors associated with submicroscopic carriage, and assessed the natural history of submicroscopic malaria over 4 weeks. RESULTS: Among 6076 participants, P. falciparum prevalences by RDT, microscopy, and PCR were 9%, 9%, and 28%, respectively, with roughly two-thirds of PCR-positive individuals harboring submicroscopic infection. Adult status, female sex, dry season months, screened windows, and bed net use were associated with submicroscopic carriage. Among 15 villages encompassing 80% of participants, the proportion of submicroscopic carriers increased with decreasing village-level malaria prevalence. Over 4 weeks, 23% of submicroscopic carriers (61 of 266) became RDT positive, with half exhibiting symptoms, while half (133 of 266) were no longer parasitemic at the end of 4 weeks. Progression to RDT-positive patent malaria occurred more frequently in villages with higher malaria prevalence. CONCLUSIONS: Microheterogeneity in transmission observed at the village level appears to affect both the size of the submicroscopic reservoir and the likelihood of submicroscopic carriers developing patent malaria in coastal Tanzania.

The effect of an anti-malarial herbal remedy, Maytenus senegalensis, on electrocardiograms of healthy Tanzanian volunteers.

BACKGROUND: The emergence of resistance to artemisinin-based combination therapy necessitates the search for new, more potent antiplasmodial compounds, including herbal remedies. The whole extract of Maytenus senegalensis has been scientifically investigated for potential biological activities both in vitro and in vivo, demonstrating strong antimalarial activity. However, there is a lack of data on the electrocardiographic effects of M. senegalensis in humans, which is a crucial aspect in the investigation of malaria treatment. Assessing the electrocardiographic effects of M. senegalensis is essential, as many anti-malarial drugs can inadvertently prolong the QT interval on electrocardiograms. Therefore, the study's objective was to evaluate the electrocardiographic effects of M. senegalensis in healthy adult volunteers. METHODS: This study is a secondary analysis of an open-label single-arm dose escalation. Twelve healthy eligible Tanzanian males, aged 18 to 45, were enrolled in four study dose groups. A single 12-lead electrocardiogram (ECG) was performed at baseline and on days 3, 7, 14, 28, and 56. RESULTS: No QTcF adverse events occurred with any drug dose. Only one volunteer who received the highest dose (800 mg) of M. senegalensis experienced a moderate transient change (△QTcF > 30 ms; specifically, the value was 37 ms) from baseline on day 28. There was no difference in maximum QTcF and maximum △QTcF between volunteers in all four study dose groups. CONCLUSIONS: A four-day regimen of 800 mg every 8 h of M. senegalensis did not impact the electrocardiographic parameters in healthy volunteers. This study suggests that M. senegalensis could be a valuable addition to malaria treatment, providing a safer alternative and potentially aiding in the battle against artemisinin-resistant malaria. The results of this study support both the traditional use and the modern therapeutic potential of M. senegalensis. They also set the stage for future research involving larger and more diverse populations to explore the safety profile of M. senegalensis in different demographic groups. This is especially important considering the potential use of M. senegalensis as a therapeutic agent and its widespread utilization as traditional medicine. Trial registration ClinicalTrials.gov, NCT04944966. Registered 30 June 2021-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT04944966?term=kamaka&draw=2&rank=1.

Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in mainland Tanzania, 2018.

BACKGROUND: The use of artemisinin-based combination therapy (ACT) is recommended by the World Health Organization for the treatment of uncomplicated falciparum malaria. Artemether-lumefantrine (AL) is the most widely adopted first-line ACT for uncomplicated malaria in sub-Saharan Africa (SSA), including mainland Tanzania, where it was introduced in December 2006. The WHO recommends regular assessment to monitor the efficacy of the first-line treatment specifically considering that artemisinin partial resistance was reported in Greater Mekong sub-region and has been confirmed in East Africa (Rwanda and Uganda). The main aim of this study was to assess the efficacy and safety of AL for the treatment of uncomplicated falciparum malaria in mainland Tanzania. METHODS: A single-arm prospective anti-malarial drug efficacy trial was conducted in Kibaha, Mlimba, Mkuzi, and Ujiji (in Pwani, Morogoro, Tanga, and Kigoma regions, respectively) in 2018. The sample size of 88 patients per site was determined based on WHO 2009 standard protocol. Participants were febrile patients (documented axillary temperature ≥ 37.5 °C and/or history of fever during the past 24 h) aged 6 months to 10 years. Patients received a 6-dose AL regimen by weight twice a day for 3 days. Clinical and parasitological parameters were monitored during 28 days of follow-up to evaluate the drug efficacy and safety. RESULTS: A total of 653 children were screened for uncomplicated malaria and 349 (53.7%) were enrolled between April and August 2018. Of the enrolled children, 345 (98.9%) completed the 28 days of follow-up or attained the treatment outcomes. There were no early treatment failures, but recurrent infections were higher in Mkuzi (35.2%) and Ujiji (23%). By Kaplan-Meier analysis of polymerase chain reaction (PCR) uncorrected adequate clinical and parasitological response (ACPR) ranged from 63.4% in Mkuzi to 85.9% in Mlimba, while PCR-corrected ACPR on day 28 varied from 97.6% in Ujiji to 100% in Mlimba. The drug was well tolerated; the commonly reported adverse events were cough, runny nose, and abdominal pain. No serious adverse event was reported. CONCLUSION: This study showed that AL had adequate efficacy and safety for the treatment of uncomplicated falciparum malaria. The high number of recurrent infections were mainly due to new infections, indicating the necessity of utilizing alternative artemisinin-based combinations, such as artesunate amodiaquine, which provide a significantly longer post-treatment prophylactic effect.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

BACKGROUND: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. METHODS: Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. RESULTS: Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. CONCLUSION: Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in mainland Tanzania, 2019.

BACKGROUND: Artemisinin-based combination therapy (ACT) has been a major contributor to the substantial reductions in global malaria morbidity and mortality over the last decade. In Tanzania, artemether-lumefantrine (AL) was introduced as the first-line treatment for uncomplicated Plasmodium falciparum malaria in 2006. The World Health Organization (WHO) recommends regular assessment and monitoring of the efficacy of the first-line treatment, specifically considering that artemisinin resistance has been confirmed in the Greater Mekong sub-region. This study's main aim was to assess the efficacy and safety of AL for treating uncomplicated P. falciparum malaria in Tanzania. METHODS: This was a single-arm prospective antimalarial drug efficacy trial conducted in four of the eight National Malaria Control Programme (NMCP) sentinel sites in 2019. The trial was carried out in outpatient health facilities in Karume-Mwanza region, Ipinda-Mbeya region, Simbo-Tabora region, and Nagaga-Mtwara region. Children aged six months to 10 years with microscopy confirmed uncomplicated P. falciparum malaria who met the inclusion criteria were recruited based on the WHO protocol. The children received AL (a 6-dose regimen of AL twice daily for three days). Clinical and parasitological parameters were monitored during follow-up over 28 days to evaluate drug efficacy. RESULTS: A total of 628 children were screened for uncomplicated malaria, and 349 (55.6%) were enrolled between May and September 2019. Of the enrolled children, 343 (98.3%) completed the 28-day follow-up or attained the treatment outcomes. There were no early treatment failures; recurrent infections during follow-up were common at two sites (Karume 29.5%; Simbo 18.2%). PCR-corrected adequate clinical and parasitological response (ACPR) by survival analysis to AL on day 28 of follow-up varied from 97.7% at Karume to 100% at Ipinda and Nagaga sites. The commonly reported adverse events were cough, skin pallor, and abdominal pain. The drug was well tolerated, and no serious adverse event was reported. CONCLUSION: This study showed that AL had adequate efficacy and safety for the treatment of uncomplicated falciparum malaria in Tanzania in 2019. The high recurrent infections were mainly due to new infections, highlighting the potential role of introducing alternative artemisinin-based combinations that offer improved post-treatment prophylaxis, such as artesunate-amodiaquine (ASAQ).

Evaluation of Malaria Rapid Diagnostic Test Performance and pfhrp2 Deletion in Tanzania School Surveys, 2017.

As part of malaria nationwide monitoring and evaluation initiatives, there is an increasing trend of incorporating malaria rapid diagnostic tests (mRDTs) in surveys conducted within primary schools to detect malaria parasites. However, mRDTs based on the detection of histidine-rich protein 2 (HRP2) are known to yield false-positive results due to persistent antigenemia, and false-negative results may result from low parasitemia or Plasmodium falciparum hrp2/3 gene deletion. We evaluated diagnostic performance of an HRP2 and pan-parasite lactate dehydrogenase (HRP2/pLDH) mRDT against polymerase chain reaction (PCR) for detection of P. falciparum among 17,051 primary school-age children from eight regions of Tanzania in 2017. According to PCR, the prevalence of P. falciparum was 19.2% (95% CI: 18.6-19.8). Using PCR as reference, the sensitivity and specificity of mRDT was 76.2% (95% CI: 74.7-77.7) and 93.9% (95% CI: 93.5-94.3), respectively. Test agreement was lowest in low transmission areas, where true-positive mRDTs were outnumbered by false-negatives due to low parasitemia. Discordant samples (mRDT-negative but PCR-positive) were screened for pfhrp2/3 deletion by real-time PCR. Among those with a parasite density sufficient for analysis, pfhrp2 deletion was confirmed in 60 samples, whereas pfhrp3 deletion was confirmed in two samples; one sample had both pfhrp2 and pfhrp3 deletions. The majority of samples with gene deletions were detected in the high-transmission Kagera region. Compared with mRDTs, PCR and other molecular methods offer increased sensitivity and are not affected by pfhrp2/3 deletions, making them a useful supplement to mRDTs in schools and other epidemiological surveys.

Efficacy of artesunate-amodiaquine for treatment of uncomplicated Plasmodium falciparum malaria in mainland Tanzania.

BACKGROUND: Diversification of artemisinin-based combination therapy (ACT) is suggested as one of the strategies that can be used to contain artemisinin resistance. Artesunate-amodiaquine (ASAQ) is one of the artemisinin-based combinations that can be used in the diversification strategy as an alternative first-line treatment for uncomplicated malaria in mainland Tanzania. There is however limited data on the efficacy of ASAQ in mainland Tanzania. This study assessed the efficacy of ASAQ for treatment of uncomplicated Plasmodium falciparum malaria in selected sentinel sites for therapeutic efficacy studies in mainland Tanzania. METHODS: Between December 2018 and March 2020, children aged between 6 months and 10 years, attending at Nagaga, Mkuzi, and Mlimba primary health facilities, and with suspected uncomplicated malaria infection were screened for eligibility to participate in the study. Malaria infection was screened using microscopy. Children with uncomplicated P. falciparum monoinfection and who fulfilled all other inclusion criteria, and had none of the exclusion criteria, according to the World Health Organization (WHO) guidelines, were treated with ASAQ. Follow-up visits were scheduled on days 0, 1, 2, 3, 7, 14, 21, and 28 or on any day of recurrent infection for clinical and laboratory assessment. Polymerase chain reaction (PCR)-corrected cure rate on day 28 was the primary outcome. RESULTS: A total of 264 children, 88 in each of the three study sites (Mlimba, Mkuzi and Nagaga health facilities) were enrolled and treated with ASAQ. The ASAQ PCR-corrected cure rate was 100% at all the three study sites. None of the participants had early treatment failure or late clinical failure. Furthermore, none of the participants had a serious adverse event. CONCLUSION: ASAQ was highly efficacious for the treatment of uncomplicated P. falciparum malaria in mainland Tanzania, therefore, it can be deployed as an alternative first-line treatment for uncomplicated malaria as part of diversification strategy to contain the spread of partial artemisinin resistance in the country.

Microsatellites reveal high polymorphism and high potential for use in anti-malarial efficacy studies in areas with different transmission intensities in mainland Tanzania.

BACKGROUND: Tanzania is currently implementing therapeutic efficacy studies (TES) in areas of varying malaria transmission intensities as per the World Health Organization (WHO) recommendations. In TES, distinguishing reinfection from recrudescence is critical for the determination of anti-malarial efficacy. Recently, the WHO recommended genotyping polymorphic coding genes, merozoite surface proteins 1 and 2 (msp1 and msp2), and replacing the glutamate-rich protein (glurp) gene with one of the highly polymorphic microsatellites in Plasmodium falciparum to adjust the efficacy of antimalarials in TES. This study assessed the polymorphisms of six neutral microsatellite markers and their potential use in TES, which is routinely performed in Tanzania. METHODS: Plasmodium falciparum samples were obtained from four TES sentinel sites, Kibaha (Pwani), Mkuzi (Tanga), Mlimba (Morogoro) and Ujiji (Kigoma), between April and September 2016. Parasite genomic DNA was extracted from dried blood spots on filter papers using commercial kits. Genotyping was done using six microsatellites (Poly-α, PfPK2, TA1, C3M69, C2M34 and M2490) by capillary method, and the data were analysed to determine the extent of their polymorphisms and genetic diversity at the four sites. RESULTS: Overall, 83 (88.3%) of the 94 samples were successfully genotyped (with positive results for ≥ 50.0% of the markers), and > 50.0% of the samples (range = 47.6-59.1%) were polyclonal, with a mean multiplicity of infection (MOI) ranging from 1.68 to 1.88 among the four sites. There was high genetic diversity but limited variability among the four sites based on mean allelic richness (RS = 7.48, range = 7.27-8.03, for an adjusted minimum sample size of 18 per site) and mean expected heterozygosity (He = 0.83, range = 0.80-0.85). Cluster analysis of haplotypes using STRUCTURE, principal component analysis, and pairwise genetic differentiation (FST) did not reveal population structure or clustering of parasites according to geographic origin. Of the six markers, Poly-α was the most polymorphic, followed by C2M34, TA1 and C3M69, while M2490 was the least polymorphic. CONCLUSION: Microsatellite genotyping revealed high polyclonality and genetic diversity but no significant population structure. Poly-α, C2M34, TA1 and C3M69 were the most polymorphic markers, and Poly-α alone or with any of the other three markers could be adopted for use in TES in Tanzania.

Sexual and reproductive health among men with genital schistosomiasis in southern Tanzania: A descriptive study.

Male genital schistosomiasis (MGS) is a significantly neglected condition, and its consequences often receive inadequate attention. The disease is suggested to cause schistosomiasis-induced sexual and reproductive health problems among males. The study was conducted to investigate the prevalence of MGS, sexual and reproductive health problems that could be caused by MGS among adult males in Mtama district. A community-based cross-sectional study using quantitative methods was carried out among males aged ≥ 18 years in selected households. Semen and urine samples were collected from each participant to establish the prevalence of MGS and urogenital schistosomiasis respectively. Semen quality was macroscopically and microscopically assessed. Urine samples were analyzed using filtration technique. A structured questionnaire interview was carried out to collect socio-demographic data, sexual and reproductive health information. Descriptive statistics were used to provide a summary of each variable. The prevalence (proportions) were presented in percentages and their respective 95% confidence intervals. A total of 223 adult males participated in this study. The prevalence of MGS and urogenital schistosomiasis were 5.8% (95% CI; 3.1%-9.0%) and 22.4% (95% CI; 16.6%-27.8%) respectively. The prevalence of Schistosoma haematobium eggs in semen was found high among young adults 12/129 (9.3%, 95% CI; 4.9%-15.7%), who never attended to school 6/35 (17.1%, 95% CI; 6.6%-33.6%), petty traders 4/26 (15.4%, 95% CI; 4.4%-34.9%), never impregnated woman 9/70 (12.9%, 95% CI: 6.6%-33.6%), experienced pain during ejaculation 4/17 (23.5%, 95% CI; 4.9%-15.7%), and with brownish semen 2/5 (40%, 95% CI; 4.9%-15.7%). According to the findings, MGS, like urogenital schistosomiasis, is prevalent in southern Tanzania. The disease is prevalent among males with some reproductive and sexual issues. This highlight the need for more research to investigate the association of MGS and male reproductive and sexual health for improved health services among males.

Trends of Plasmodium falciparum molecular markers associated with resistance to artemisinins and reduced susceptibility to lumefantrine in Mainland Tanzania from 2016 to 2021.

BACKGROUND: Therapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemisinin-based combination therapy (ACT). This study assessed the trends of molecular markers of artemisinin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021. METHODS: A total of 2,015 samples were collected during TES of artemether-lumefantrine at eight sentinel sites (in Kigoma, Mbeya, Morogoro, Mtwara, Mwanza, Pwani, Tabora, and Tanga regions) between 2016 and 2021. Photo-induced electron transfer polymerase chain reaction (PET-PCR) was used to confirm presence of malaria parasites before capillary sequencing, which targeted two genes: Plasmodium falciparum kelch 13 propeller domain (k13) and P. falciparum multidrug resistance 1 (pfmdr1). RESULTS: Sequencing success was ≥ 87.8%, and 1,724/1,769 (97.5%) k13 wild-type samples were detected. Thirty-seven (2.1%) samples had synonymous mutations and only eight (0.4%) had non-synonymous mutations in the k13 gene; seven of these were not validated by the WHO as molecular markers of resistance. One sample from Morogoro in 2020 had a k13 R622I mutation, which is a validated marker of artemisinin partial resistance. For pfmdr1, all except two samples carried N86 (wild-type), while mutations at Y184F increased from 33.9% in 2016 to about 60.5% in 2021, and only four samples (0.2%) had D1246Y mutations. pfmdr1 haplotypes were reported in 1,711 samples, with 985 (57.6%) NYD, 720 (42.1%) NFD, and six (0.4%) carrying minor haplotypes (three with NYY, 0.2%; YFD in two, 0.1%; and NFY in one sample, 0.1%). Between 2016 and 2021, NYD decreased from 66.1% to 45.2%, while NFD increased from 38.5% to 54.7%. CONCLUSION: This is the first report of the R622I (k13 validated mutation) in Tanzania. N86 and D1246 were nearly fixed, while increases in Y184F mutations and NFD haplotype were observed between 2016 and 2021. Despite the reports of artemisinin partial resistance in Rwanda and Uganda, this study did not report any other validated mutations in these study sites in Tanzania apart from R622I suggesting that intensified surveillance is urgently needed to monitor trends of drug resistance markers and their impact on the performance of ACT.

Accuracy and precision of dried urine spot method for the detection of Schistosoma mansoni circulating cathodic antigens in resource-limited settings.

BACKGROUND: The World Health Organization recommends the use of Schisto point-of-care circulating cathodic antigens (Schisto POC-CCA) for screening of Schistosoma mansoni as it offers better sensitivity than microscopy. However, there are limitation facing the use of this method including timely availability of the test cassettes. The aim of this study was to determine the reliability of dried urine spot (DUS) method for collection of urine and detection of S. mansoni using Schisto POC-CCA cassettes in a resource-limited settings. METHODS: A cross-sectional study was conducted between October and November 2022 among 250 primary school children in Sengerema District, northwestern Tanzania. S. mansoni CCA was detected in filter paper-based DUS, liquid urine using DUS Schisto POC-CCA (index), and direct urine Schisto POC-CCA (comparator) methods respectively. S. mansoni eggs in stool were detected using duplicate Kato-Katz (KK) method. The measures of accuracy were computed and compared between the index and comparator methods. The strength of agreement between inter-raters precisions was tested using Cohen's kappa (k). RESULTS: This study revealed S. mansoni prevalence rates of 28.8%, 54.0% and 50.8% by duplicate KK, direct urine Schisto POC-CCA and DUS Schisto POC-CCA methods respectively. The mean intensity of infection among infected participants was 86.3 eggs per gram of stool (EPG) ranging from 12.0 EPG to 824.0 EPG. The sensitivity of DUS Schisto POC-CCA and direct urine Schisto POC-CCA was 94.44% (95% CI: 89.15-99.74%) and 97.22% (95% CI: 93.43-100.00%) respectively. The DUS Schisto POC-CCA method had slightly higher specificity (66.85%) than direct urine Schisto POC-CCA method (63.48%). The accuracy of the DUS Schisto POC-CCA was found to be slightly high (74.80%, 95% CI: 68.94-79.06%) compared to that of direct urine Schisto POC-CCA (73.20%, 95% CI: 67.25-78.59%). There was good agreement between two laboratory technologists who performed the DUS Schisto POC-CCA method on similar samples (k = 0.80, 95% CI: 0.59-0.95). CONCLUSIONS: The DUS Schisto POC-CCA method had comparable S. mansoni detection accuracy to direct urine Schisto POC-CCA. This suggests that the method could be a potential alternative to direct urine Schisto POC-CCA for screening S. mansoni in resource-limited situations.

Evaluation of the DN-Mini (miniaturized double net) trap for sampling host-seeking Anopheles mosquitoes in malaria-endemic villages of southern Tanzania.

BACKGROUND: Surveillance of malaria vectors is crucial for assessing the transmission risk and impact of control measures. Human landing catches (HLC) directly estimate the biting rates but raise ethical concerns due to the exposure of volunteers to mosquito-borne pathogens. A common alternative is the CDC-light trap, which is effective for catching host-seeking mosquitoes indoors but not outdoors. New, exposure-free methods are needed for sampling mosquitoes indoors and outdoors in ways that reflect their natural risk profiles. The aim of this study was therefore to evaluate the efficacy of the miniaturized double net trap (DN-Mini) for sampling host-seeking mosquitoes in south-eastern Tanzania, where malaria transmission is dominated by Anopheles funestus. METHODS: Adult mosquitoes were collected from 222 randomly selected houses across three villages (74 per village) in Ulanga district, south-eastern Tanzania, using the DN-Mini traps, CDC-Light traps, and Prokopack aspirators. First, we compared CDC-light and DN-Mini traps for collecting indoor host-seeking mosquitoes, while Prokopack aspirators were used for indoor-resting mosquitoes. Second, we deployed the DN-Mini and Prokopack aspirators to collect host-seeking and resting mosquitoes indoors and outdoors. Generalized linear mixed models (GLMM) with a negative binomial distribution were used to compare the effectiveness of the traps for catching different mosquito species. RESULTS: The DN-Mini was 1.53 times more efficient in collecting An. funestus indoors (RR = 1.53, 95% CI: 1.190-1.98) compared to the CDC-Light trap. However, for Anopheles arabiensis, the DN-Mini caught only 0.32 times as many mosquitoes indoors as the CDC-Light traps (RR = 0.32, 95% CI: 0.183-0.567). Both An. funestus and An. arabiensis were found to be more abundant indoors than outdoors when collected using the DN-Mini trap. Similarly, the Prokopack aspirator was greater indoors than outdoors for both An. funestus and An. arabiensis. CONCLUSION: The DN-Mini outperformed the CDC-light trap in sampling the dominant malaria vector, An. funestus species, but was less effective in capturing An. arabiensis, and for both vector species, the biting risk was greater indoors than outdoors when measured using the DN-Mini trap. These findings highlight the importance of selecting appropriate trapping methods based on mosquito species and behaviors.

Antibody-Dependent Respiratory Burst against Plasmodium falciparum Merozoites in Individuals Living in an Area with Declining Malaria Transmission.

Malaria transmission intensity affects the development of naturally acquired immunity to malaria. An absolute correlate measure of protection against malaria is lacking. However, antibody-mediated functions against Plasmodium falciparum correlate with protection against malaria. In children, antibody-mediated functions against P. falciparum decline with reduced exposure. It is unclear whether adults maintain antibody-mediated functions as malaria transmission declines. This study assessed antibody-dependent respiratory burst (ADRB) in individuals from an area with declining malaria transmission. In an age-matched analysis, we compare ADRB activity during high versus low malaria transmission periods. Age significantly predicted higher ADRB activity in the high (p < 0.001) and low (p < 0.001) malaria transmission periods. ADRB activity was higher during the high compared to the low malaria transmission period in older children and adults. Only older adults during the high malaria transmission period had their median ADRB activity above the ADRB cut-off. Ongoing P. falciparum infection influenced ADRB activity during the low (p = 0.01) but not the high (p = 0.29) malaria transmission period. These findings propose that naturally acquired immunity to P. falciparum is affected in children and adults as malaria transmission declines, implying that vaccines will be necessary to induce and maintain protection against malaria.